Determination of β-Galactooligosaccharides (GOS) in Infant Formula and Adult Nutritionals: Single-Laboratory Validation, First Action 2021.01

Abstract Background β-Galactooligosaccharides (GOS) are typically used in infant formula and adult nutritionals as a source of nondigestible oligosaccharides, which may bring beneficial effects through modulation of the gut microbiota. However, suitable methods for the determination of GOS in products with a high background of lactose do not exist. Objective The aim of this work was to develop a method suitable for the determination of GOS in infant formula and adult nutritionals and demonstrate suitability through single laboratory validation. Methods Reducing oligosaccharides are labeled with 2-aminobenzamide (2AB), separated by hydrophilic interaction LC, and determined assuming that all oligosaccharides give an equimolar response in the detector. The same sample is analyzed a second time after treatment with β-galactosidase to remove GOS. The difference in the determined oligosaccharides between the two measurements will be the GOS content of the sample. The method was validated in a single laboratory on infant formula and adult nutritionals. Results Recoveries were in the range 91.5–102%, relative standards of deviation (RSDr) were in the range 0.7–5.99%, and one sample had an RSDr of 8.30%. Except for the one sample with an RSDr of 8.30%, the performance is within the requirements outlined in the Standard Method Performance Requirements, which specifies recoveries in the range 90–110% and RSDr of below 6%. Conclusions The method is suitable for the determination of GOS in infant formula and adult nutritionals. Highlights A method has been developed which is suitable for the determination of GOS in products with a high background concentration of lactose (infant fromula and adult nutritionals). The method does not require access to the GOS ingredient used for the production of the finished product. It is also possible to separately quantify the amount of GOS containing three or more monomeric units in order to support dietary fibre analysis.

There are few methods available for the analysis of GOS in food products. In 2002 de Slegte (19) published a validated method. The method employs high-performance anion exchange chromatography with pulsed amperometric detection (HPEAC-PAD) to determine glucose, galactose, and lactose in a product. The product is then hydrolyzed using a b-galactosidase to transform all the GOS and lactose to glucose and galactose. The increase in galactose after hydrolysis is used to determine the GOS content after correction for the galactose released from lactose. This method has been evaluated in an inter-laboratory study and is the AOAC Official Method SM 2001.02 (20). However, the method has a couple of weak points. Since the method is based on analyzing only the released galactose, the user needs to know the relative amounts of galactose to glucose in the GOS to correct for the non-analyzed glucose. Furthermore, because the method relies on measuring the difference in released galactose before and after hydrolysis, it is not well adapted to analyzing products containing high background concentrations of galactose or lactose, such as those found in infant formula. The method was improved by Hui et al. in 2018 (21), to encompass the analysis of both glucose and galactose and thus avoid the need for knowledge of the glucose to galactose ratio of the GOS ingredient. However, the basic principles of the method remain the same, and the updated method remains ill-suited to the analysis of GOS in samples with a high background of lactose.
In 2010 Albrecht et al. (22) demonstrated that GOS could be well separated and analyzed using capillary electrophoresis. The authors achieved promising results with products, including infant formula, but did not attempt method validation. In 2014 Austin et al. (23) developed a method for the analysis of GOS by hydrophilic interaction LC (HILIC). The method was based on the approach for the analysis of glycoprotein glycans described by Guile et al. (24). Oligosaccharides were extracted from the sample in water, and then labeled with 2-aminobenzamide (2AB). After labeling, maltodextrins were removed by treatment with amyloglucosidase; then the labeled oligosaccharides were separated by HILIC and the 2AB tag was detected using a fluorescence detector (FLD). Since it is the 2AB tag that is detected, and each oligosaccharide carries only a single tag, the detector signal is proportional to the molar concentration of the oligosaccharides. This enables the quantification of GOS without the need for analytical standards for every individual oligosaccharide present. The method performance was assessed through single-laboratory validation (SLV) and the results were promising. However, a major issue was the nonspecificity of the method, since the 2AB tag will label any reducing oligosaccharide with an aldose at the reducing end. The work described here is based on that method. To introduce specificity for GOS, two analyses are performed, one with and one without treatment with b-galactosidase. This once again has the disadvantage that we are measuring GOS by difference. However, since the method targets oligosaccharides, there is no significant overabundance of background that needs to be subtracted (as in the case of methods targeting galactose after hydrolytic release), so the results are more reliable.  Table 2. The kit contains five products described as placebo products and 14 products described as fortified products. Four of the fortified products were found to contain GOS. It was also found that one of the placebo products contained GOS. Five additional matrices were added to increase the number of samples containing GOS. All samples were stored in the original packaging in a dry place, protected from light until the moment of use.  (25). b Concentrations apply to the product as consumed (i.e., on reconstituted powders or concentrates or "as is" on ready to feed (RTF) products). Prior to analysis, all powder products were reconstituted by dissolving 25 g powder in 200 g water and all ready-to-feed (RTF) products were used as is. The in-house reference sample was analyzed without prior reconstitution.

Validation Design
(a) Calibration fit.-This was assessed by injecting calibration solutions of maltotriose (11-2100 nmol/mL) at 11 different concentrations, prepared in triplicate, and all containing the laminaritriose internal standard. The ratio of the peak areas (maltotriose/laminaritriose) was plotted against the concentration of maltotriose (since the laminaritriose concentration remained constant throughout there was no need to plot the ratio of the concentrations on the x-axis). A linear model was used to fit the data for calibration purposes. The differences between actual concentration and the concentration predicted by the calibration model were calculated and plotted against the analyte concentration.
(c) Repeatability (r) and intermediate reproducibility (iR).-These were assessed by analyzing samples (containing GOS) in duplicate on at least six different days by two different operators on two different instruments. The inhouse statistical package Q-Stat was used to calculate the SD r and SD iR using Equations 1 and 2: where: n ¼ number of (single or duplicate) determinations; x i ¼ individual result within the set of single determinations with i going from 1 to n; x i1 ,x i2 ¼ two results within the set of duplicate determination with i going from 1 to n; SD(b) ¼ SD between the means of duplicates; SD r ¼ SD for repeatability; and SD iR ¼ SD for intermediate reproducibility.
(d) LOD and LOQ.-Because the method requires the analysis of a complete profile of oligosaccharides, the detection and quantification limits depend on the GOS profile as well as the concentration of GOS in the product, making it extremely difficult to assess (except on an individual oligosaccharide basis). To demonstrate method applicability at  In addition to the spike-recovery experiments, LOD and LOQ were assessed based on the measurement of blank samples. In the SPIFAN kit 14 samples were included that did not contain GOS. Each of these was analyzed in duplicate to generate a mean and SD for each blank sample. The SDs of the blank results were combined according to Equation 1. The LOD and LOQ were then estimated using Equations 3 and 4: where GOS blk ¼ largest GOS content measured in the 14 blank samples and SD blk ¼ combined SD of all the blank samples. In addition, the LOQ for a single oligosaccharide was assessed using the maltotriose standard by measuring the S/N when low concentrations of maltotriose were injected on the system, the S/N versus concentration was plotted, and the LOQ assigned at the concentration where S/N was 10. This was assessed on two different instruments. Caution: The method employs corrosive, toxic (acute and irritant), and flammable chemicals such as acetic acid, sodium hydroxide, formic acid, ammonia solution, and acetonitrile. Amyloglucosidase is a health hazard and ammonia solution is also dangerous for the environment. Sodium cyanoborohydride (NaBH 3 CN) is very toxic, highly flammable, and dangerous for the environment. Wear personal protective equipment such as gloves and safety glasses. Perform all manipulations under a fume hood. Refer to the materials safety data sheets, take appropriate additional safety precautions for handling, and ensure waste disposal is in accordance with local requirements.

A. Principle
Samples are reconstituted in water and oligosaccharides present in samples are extracted at 70 C. Duplicate aliquots of the diluted sample are taken and both are treated with amyloglucosidase to hydrolyze any maltooligosaccharides present (Assay 1). One aliquot is also treated with b-galactosidase (Assay 2) to hydrolyze all the GOS present. An internal standard (laminaritriose) is added to both aliquots and the oligosaccharides are fluorescently labeled with 2-aminobenzamide (2AB). Labeled extracts are diluted with acetonitrile prior to injection on an ultra high perfromance liquid chromatography (UHPLC) system equipped with a fluorescence detector (FLD) and a hydrophilic interaction LC (HILIC) analytical column. The analytes are separated using a gradient of aqueous ammonium formate in acetonitrile and detected with an FLD. An external maltotriose calibration curve is prepared in the same way as the samples but without enzymatic treatment. Since it is the 2AB label that is detected, each oligosaccharide has an equivalent molar response. The maltotriose calibration curve can thus be used to determine the molar concentrations of the oligosaccharides in the two assays. It is then necessary to know the MW of each signal in the chromatogram to convert the molar concentrations to mass concentrations. This can be done by coupling a mass spectrometer (but once a GOS ingredient profile has been characterized by HILIC-FLD-MS, future samples can be analyzed without the MS). They may also be estimated by comparing the relative retention time (RRT) of the oligosaccharide against that of a dextran ladder, similar to the approach used for glycan analysis (24). The GOS content is obtained by subtracting the oligosaccharide (OS) content obtained in Assay 2 from the OS content obtained in Assay 1.         Table 2021.01C for quantities). Mix the solution using a vortex mixer. Weigh the amount of 2-aminobenzamide (2AB) and sodium cyanoborohydride (NaBH 3 CN) (see Table 2021.01C) in a 10 mL glass tube, and then add the corresponding volume of 30% acetic acid in DMSO. Mix (vortex) and use an ultrasonic bath for complete dissolution (about 10 min). No. 10102857001, also available from Roche Diagnostics, has also been tested and found to be suitable. This enzyme is already in suspension (140 U/mL) and can be diluted with 0.2 M sodium acetate buffer pH 4.5 in order to produce a working concentration (60 U/mL). When enzymes from another source are used it is imperative to ensure the enzymes employed will completely hydrolyze any maltodextrins in the product without hydrolyzing any analytes, as well as not showing any interference in the chromatogram. This can be checked by performing an analysis with maltodextrin as a sample, a GOS ingredient as a sample, and running a blank with the amyloglucosidase only.

C. Chemicals and Reagents
(h) b-Galactosidase solution (4000 U/mL).-Use the solution as is.
Note: For the development and validation of this method, the b-galactosidase E-BGLAN, available from Megazyme, was used. When enzyme from another source is used it is imperative to ensure the enzyme employed will completely hydrolyze the GOS without hydrolyzing any other oligosaccharides that may be present in the sample.

F. Preparation of Standards
Prepare a six-level calibration curve by diluting the maltotriose stock solution as described in Table 2021.01D, using volumetric flasks made up to the final volume with water. Cool the solution to room temperature. (b) For reconstituted products (as prepared above), or products that are sold as RTF, weigh an amount of sample (m) containing a maximum of 40 mg GOS, but not more than 5 g of sample, into a 25 mL (V) volumetric flask and make up to the mark with water. (c) For analysis of homogeneous powder products without prior reconstitution, weigh an amount of sample (m) containing a maximum of 80 mg GOS, but not more than 1.1 g of powder, into a 50 mL (V) volumetric flask. Add water (30 mL) and heat at 70 C with constant agitation for 25 min. Cool to room temperature and complete to the mark with water.

H. Chromatographic Conditions
The UHPLC system is equipped with an Acquity UPLC BEH Glycan column (2.1 mm Â 150 mm, 1.7 mm). The column is held at 25 6 1 C and the injection volume is 2 mL. The analytes are separated using the gradient described in Table 2021.01E and are detected by means of an FLD tuned at the following wavelengths: excitation k ¼ 330 nm and emission k ¼ 420 nm.

I. System Suitability Check
Before starting an analysis allow the chromatographic system to equilibrate and the FLD to warm up (if necessary) under the initial conditions, for at least 15 min. Let the derivatized standard and sample solutions equilibrate to the autosampler temperature before making any injections.

J. Calibration and Calculations
It is recommended to use bracketed calibration, injecting three standards followed by a maximum of 10 samples, then three standards, etc. For example, inject standards at levels 1, 3 and 5, then 10 samples, then standards at levels 2, 4 and 6, then 10 samples, then standards 1, 3, 5, etc. Use the instrument software to plot a six-point standard curve of "instrument response for maltotriose/instrument response for laminaritriose" against "concentration of maltotriose" in the standard (in mmol/mL). Fit a linear model to the data including the origin as a point (but not forced through the origin).
When integrating the peaks in the chromatogram it is important to make an estimate of the S/N of the smaller peaks. Only include peaks with an S/N of 10 or greater in the calculations. Smaller peaks cannot be quantified accurately and introduce inaccuracies in the measurement if included. Do not integrate the lactose peak or the maltose peak (if present). In Assay 2, a peak  may appear in the region that was covered by the lactose signal in Assay 1; do not integrate this peak. Use the standard curve to calculate the molar concentration (in mmol/mL) of each oligosaccharide in the chromatogram (C m ) without b-galactosidase treatment (Assay 1) and calculate the total oligosaccharides in that sample using Equation 5. Then use the standard curve to calculate the molar concentration (in mmol/mL) of each oligosaccharide in the chromatogram (C m ) after b-galactosidase treatment (Assay 2) and calculate the total oligosaccharides in that sample according to Equation 6. Then calculate the GOS content of the sample using Equation 7. If it is desired to calculate the dietary fiber content of the GOS, exclude the disaccharides from the calculations in Equations 5 and 6: where: C TOS ¼ total concentration of oligosaccharides in the untreated sample (in g/100 g); C B ¼ total concentration of oligosaccharides in the enzyme-treated sample (in g/100 g); C GOS ¼ total concentration of GOS in the sample (in g/100 g); C m ¼ molar concentration of each individual oligosaccharide in the sample (in mmol/mL); MW ¼ MW of each individual oligosaccharide in the sample, either estimated from the glucose unit (GU) value (Annex B) or measured by MS (Annex C); V ¼ volume to which the original sample weight was diluted (in mL); m ¼ weight of sample diluted to volume (V) (in g); and 0.0001 ¼ factor to convert the result from mg/g to g/100 g.

K. Annex A-Assessment of Maltotriose Concentration
In general, the manufacturer's certificate of analysis (CoA) provides sufficient information to accurately calculate the concentration of the maltotriose stock solution (having corrected for both the moisture content and the purity of the standard). However, in some cases the CoA may not be available, or the data provided may be inaccurate. In these cases, the purity and moisture content of the maltotriose standard needs to be assessed. (2) Glass tubes (10 mL) with rubber stoppers for air-tight sealing.
(3) Add 5 g solvent mixture and dissolve the sample (use a vortex mixer and/or sonic bath).
(4) Weigh 500 mg solvent mixture (without sample) in a syringe and inject it into the coulometer to determine the moisture content of the solvent.
(5) Repeat the analysis of the blank solvent mixture two more times, to have a total of three measurements.
(6) Weigh 500 mg sample solution in a syringe and inject into the coulometer to determine the moisture content of the sample þ solvent.  The standard is prepared and injected on the chromatographic system as described in the main method. The chromatogram is then assessed for signals in addition to the ones expected (maltotriose and the internal standard, laminaritriose). The peak areas are measured for all peaks except for laminaritriose and assigned a corresponding molecular mass depending on the identity of the signal [180 for glucose, 342 for disaccharides composed of two hexoses (Hex 2), 504 for Hex 3 (including maltotriose), 666 for Hex 4, 828 for Hex 5, 990 for Hex 6, 1152 for Hex 7; see Figure 2021.01A]. The peak area of maltotriose multiplied by its molecular mass (504) is then divided by the sum of all the peak areas multiplied by their corresponding masses to calculate the purity of maltotriose. This is best illustrated by an example.
An example data set is shown in Table 2021.01F; taking these data the purity of the maltotriose would be calculated as shown in Equation 8. (8) where: P M3 (%) ¼ purity of maltotriose in %; A M3 ¼ peak area of maltotriose; MW M3 ¼ MW of maltotriose in g/mol; A Mi ¼ peak area of signal with i hexose units; and MW Mi ¼ MW of oligosaccharide with i hexose units in g/mol. If the measured moisture and purity are in good agreement with the manufacturer's CoA (each 62 g/100 g), it is recommended to use the data provided on the manufacturer's CoA. If the difference is larger it is recommended to use the measured moisture and purity or to use a different batch of maltotriose.

L. Annex B-Determination of Molecular Weight Using the Dextran Ladder
To determine the MW of each signal it is possible to calibrate the column using the dextran ladder. The dextran oligosaccharides elute from the smallest to the largest. Isomaltose is the first signal and is composed of 2 GU, and it is therefore assigned a GU value of 2; the next oligosaccharide of the dextran ladder has a GU of 3, and so on (Figure 2021.01B).
Determine the RRT of each of the dextran signals compared to the internal standard according to Equation 9. Then make a plot of GU against RRT and fit a third-order polynomial (y ¼ ax 3 þ bx 2 þ cx þ d). For each signal in the sample chromatogram calculate the RRT in the same way as for the dextran ladder, and then assign a GU value from the polynomial. The molecular mass of each signal can then be assigned based on the GU value using Table 2021.01G. In case the mass spectrometer has insufficient sensitivity, it is possible to prepare a sample having 10 times greater GOS concentration for the purposes of peak identification only (in this case it is recommended to use the GOS ingredient). In some cases, there may be some overlap of peaks having a different MW. In these cases, the analyst should assign the MW that would be expected to result in the least error (e.g., if the MS signals of a trisaccharide and tetrasaccharide overlap, but the signal is stronger for the trisaccharide, then assign the peak the MW of a trisaccharide).

Method Development
We already developed and validated a method for the analysis of GOS in infant formula (23). The method is based on the  labeling of the oligosaccharides with a fluorescent tag, separating the labeled oligosaccharides by HILIC and then detecting the label. Assuming each labeled oligosaccharide has an equimolar response factor, the GOS content could be calculated using a surrogate oligosaccharide standard (maltotriose). The disadvantage with the approach is the lack of specificity of the method since the tag will label any oligosaccharide with an aldose at the reducing end. To overcome this issue the sample was split in two aliquots and treated with enzymes. The first enzyme, amyloglucosidase, is added to both aliquots, and removes maltodextrins, thus reducing the background oligosaccharide concentration. The second enzyme, b-galactosidase, is added only to the second aliquot and removes the GOS. The purity of the enzymes is extremely important. The amyloglucosidase should not have any activities that hydrolyze GOS, and the b-galactosidase needs to be specific for GOS. We found that amyloglucosidases commonly used for dietary fiber analysis were well suited for the selective and specific removal of maltodextrins. However, some of the enzymes introduced additional signals in blank chromatograms, which we wanted to avoid. We found that the amyloglucosidase enzymes from Roche worked well for this application. The b-galactosidases were less specific, so when selecting an enzyme, screening for side activities is recommended. We found the b-galactosidase from Megazyme (E-BGLAN, 4000 U/mL) was suitable for the application. When processing the chromatogram, all peaks need to be individually integrated, and it is important to exclude any peaks that have an S/N ratio below 10. Lactose is generally present in very large quantities resulting in a large signal in Assay 1. The lactose signal should not be integrated. After b-galactosidase treatment (Assay 2), the lactose peak is mostly removed, but a peak may remain in the window where lactose had been in Assay 1. This peak should not be integrated in Assay 2 because if present in Assay 1 it would be under the lactose and thus not quantified.
During the method validation, we used different batches of maltotriose from different suppliers to prepare the calibration curve. Since we had established a reference sample during development, we observed results biases that were linked to the batch of maltotriose used for calibration. We first suspected that the standards may have absorbed moisture during storage, so the moisture content of the standards was checked using a Karl Fischer titration and compared against the manufacturer's CoA. In general, we found the measured moisture contents of the standards were in good agreement with the manufacturer's CoA. We therefore decided to check the purity of the samples. We suspected that the most likely contaminants in maltotriose would be other maltooligosaccharides; this is easily investigated using the method developed for the GOS analysis. So, the maltotriose standards were labeled with 2AB and the oligosaccharide separated using the same chromatographic system as used for the GOS. We observed that most of the maltotriose standards contained maltose and glucose, and some signals that eluted later (Figure 2021.01A). The concentration of the signals was calculated, assuming equimolar response factors, and it was found that for some batches of maltotriose standard, the measured purity was different from the purity stated on the CoA ( Table 4). The standards delivering expected results on the reference sample were the standards for which the measured maltotriose purity was in good agreement with the CoA. It is therefore recommended that the purity of the maltotriose calibration standard be checked in a similar manner before use. When the purity measurement is within 62 g/100 g of the CoA it is recommended to use the value stated on the CoA; if the difference is greater, it is recommended to use the measured purity, or to use a different batch of maltotriose.

Method Specificity
Specificity of the method is achieved by running the analysis before and after hydrolysis with a b-galactosidase and making the difference between the two analyses. Potentially interfering oligosaccharides are not hydrolyzed by the b-galactosidase, and thus are not considered as part of the GOS. GOS is frequently found in products along with FOS. The FOS are either nonreducing oligosaccharides, and thus cannot be labeled, or they contain a reducing-end fructose, which is a ketose, and is not labeled with 2AB under the labeling conditions used. The FOS are thus not detected either before or after b-galactosidase treatment. Two of the samples used for spike-recovery experiments contained FOS and there were no interferences. Polydextrose is another oligosaccharide that may be found in GOS-containing products. When polydextrose was analyzed alone, or when spiked (at 0.4 g/100 g) into a GOS-containing sample, no interfering signals were observed. Polydextrose does not have a well-defined structure so we are not sure why this oligosaccharide is not observed. We postulate that it either has a molecular mass range beyond that of the GOS, or that there are no reducing ends susceptible to being labeled.
HMOs are emerging ingredients likely to be found in infant formula, potentially in combination with GOS. They give clear signals in the chromatogram and are not removed by treatment with b-galactosidase; thus they do not interfere with the analysis. However, HMO containing terminal galactose residues (e.g., lacto-N-tetraose (LNT) LNnT, lacto-N-hexaose (LNH), etc.) can be partially hydrolyzed, since the terminal galactose on the nonreducing end of the oligosaccharide can be removed by the b-galactosidase. This means that the oligosaccharide appears in one place in the chromatogram in Assay 1 and in a different place in Assay 2, meaning that the MW assignment will change between the two assays, and thus the GOS may be slightly overestimated. LNT and LNnT typically run in the region with the tetrasaccharides, so would be assigned as a Hex 4 in Assay 1 (Figure 1). In Assay 2 they can move to the Hex 3 region (Figure 1). It is thus important to identify the hydrolysis product in Assay 2 and assign the peak the mass of Hex 4 to correct for this shift. If using a mass spectrometer to identify the signals, this is readily done, since both the tetrasaccharide and the trisaccharide contain an N-acetylhexosamine (HexNAc) residue, which can be clearly differentiated from the oligosaccharides containing only hexose (Hex) residues. In case of doubt, the analyst can include a sample of the pure HMO and identify the retention time before and after b-galactosidase treatment.

Calibration Fit
A linear model was used to fit the data for calibration purposes ( Figure 2) and the model seems to fit the data well. The relative difference between the predicted and actual concentrations of the standards were calculated and plotted against the analyte concentration ( Figure 3); in general the predicted concentration is within 5% of the actual concentration although there are a few outliers, particularly at lower concentrations. We therefore confirm that a linear calibration model is appropriate.

LOD and LOQ
The LOD and LQQ are difficult to estimate since they depend on the profile of the GOS ingredient being used; therefore spike-recovery experiments were performed at 0.2 g/100 g, the required LOQ assigned in the SMPR (25). Spike-recovery results and the method precision at the addition level of 0.2 g/100 g were in line with the requirements of the SMPR, and thus it appears the method meets the LOQ requirements of the SMPR.
To try to estimate the LOD and LOQ, the GOS-free samples from the SPIFAN kit (14 samples) were reconstituted and analyzed in duplicate on a single day. The average measured GOS content of the 14 GOS-free samples was 0.00458 g/100 g (when considering negative results as 0). The highest GOS content measured in a blank was 0.0266 g/100 g. The combined SD for the blank results was 0.00596 g/100 g. Using the highest measured blank and the combined SD, the LOD and LOQ were estimated as: LOD ¼ 0:0266 þ 3 Â 0:00596 ¼ 0:0444 g=100 g LOQ ¼ 0:0266 þ 10 Â 0:00596 ¼ 0:0861 g=100 g We also assessed the LOD and LOQ for a single oligosaccharide on two different instruments. Low concentrations of maltotriose solution (expected to be close to the LOQ) were prepared in the same way as the standards and injected on the two instruments. The S/N was measured and plotted against concentration. The concentration of maltotriose giving rise to a peak with an S/N of 10 was defined as the LOQ. On one instrument the concentration of maltotriose resulting in an S/N of 10 was 9 nmol/mL, while on the other it was 3 nmol/mL. The combined results demonstrate that the method LOQ is lower than 0.2 g/100 g and could be close to 0.1 g/100 g. The practical LOQ will depend on the GOS profile, and at which concentration it is no longer possible to accurately quantify the smaller signals in the profile. With three different ingredients we have demonstrated that the method can accurately determine GOS at a concentration of 0.2 g/100 g, and therefore meets the LOQ requirement of the SMPR.

Precision
In order to establish which samples contained GOS, all samples from the SPIFAN kit were analyzed in duplicate on a single day, and five of the 19 samples were found to contain GOS, being:   The SMPR (25) requires that the RSD r should be less than or equal to 6%. The achieved RSD r was less than 6%, for all matrices except one (No. 23, Adult nutritional RTF), which had an RSD r of 8.3%. The RSD iR was less than 10% in all cases, suggesting that the method may be able to achieve an RSD R of less than 12% during multilaboratory testing (as required in the SMPR).

Accuracy/Trueness
Four different blank matrices were spiked at four levels with three different GOS ingredients (as described in Table 3). Recoveries were in the range 91.5-102% across the four matrices (Table 2021.01B) and the three GOS ingredients (Table 5); thus the recoveries meet the requirements set out in the SMPR. When organized by GOS ingredient (Table 5), it appears that there may be a small impact on the recovery, with GOS 2 having slightly higher recoveries than the other two GOS ingredients. Since two of the spiked samples contain FOS, and HMOs were spiked into most samples ( Table 3, Table 2021.01B), these results demonstrate that the method works well in the presence of other oligosaccharides that may be present in the sample together with the GOS.

Check-Standards
During all series of analyses check-standards (three levels) were also analyzed ( Table 6). The check-standards were prepared  with different stock solutions of maltotriose from the ones used to prepare the calibration curve. In most cases the results of the check-standards were within 65% of the expected concentration. On 2 days there were problems with the lowest check-standard; the difference from the expected value is 6.5% and 12.5%, but the results from the other check-standards and the reference sample on those 2 days were within expectations.

Impact of Approach for Peak Identification
The method requires that the molecular mass of each peak is determined, and two approaches for this are proposed. One approach is the use of a mass spectrometer, and this approach is the one that was used for the generation of the data described so far. This approach is the one most adaptable to different GOS ingredients, and once a profile has been established for a particular ingredient the specified peak masses can be applied without the need of a mass spectrometer for every analysis (since the peak pattern will remain constant). However, if a mass spectrometer is not available, it is also possible to calibrate the system using a dextran ladder. This approach is less flexible than the MS approach since the dextran ladder is used to define regions of the chromatogram where it is expected GOS having particular masses will elute. However, different GOS ingredients contain different oligosaccharides and the boundaries between where the different masses elute move between ingredients. The method is thus a compromise, with the boundaries between tri-and tetra-saccharides, between tetra-and penta-saccharides, etc., being set in a region that is not perfect for any single ingredient, but that returns acceptable data for a range of different ingredients. We compared the results using the two different approaches for peak assignment on the set of samples used for the precision study ( Table 7). The data indicate that there is excellent agreement in results using the two different approaches. However, we noticed that all samples appeared to contain the same GOS profile (that of GOS 1) and the samples covered a limited concentration range. Therefore, we did the same exercise using some of the spiked samples (Table 8) and   again the data show good alignment between the two approaches for peak assignment, at the three different spike levels and for the three different GOS ingredients.

Performance of AOAC Official Method SM 2001.02
During the method validation, the samples used for spike-recovery were also analyzed using AOAC Official Method 2001.02 (19, 20) ( Table 9). As expected, AOAC Official Method 2001.02 does not meet the requirements of the SMPR. In particular it struggles when the GOS concentrations are below 1 g/100 g, returning poor recoveries and poor precision. When concentrations are above 1 g/100 g, the spike-recovery results are generally good, but the precision of the method remains poor. The data confirm that the method is not well suited for the analysis of samples containing a high background of lactose and relatively low amounts of GOS. The new method should be recommended for analyzing such samples.

Determination of the GOS Contribution to Dietary Fiber
Codex has proposed two definitions of dietary fiber, one which only includes nondigestible polysaccharides having a degree of polymerization (DP) of 10 or more, and one which includes nondigestible polysaccharides having a DP of 3 or more (26). GOS typically have DPs below 10, and thus they would not be considered dietary fiber in countries adopting that definition. However, many countries have adopted the definition including nondigestible polysaccharides with a DP ! 3. In this case a proportion of the GOS would meet the definition of fiber, but the nondigestible disaccharide components of GOS remain outside the fiber definition. For labeling the fiber contents of a product containing GOS, it is thus useful to be able to measure the GOS having a DP of 3 and above. With this method it is possible to do so by simply excluding the disaccharides from Equations 5 and 6. There is no existing reference method for this analysis, so we were only able to assess the precision of this measurement (Table 10). For the measurement of GOS having a DP ! 3, the RSD r were quite comparable with those for GOS, being between 0.78 and 8.80% (for GOS 0.72-8.30%); however, the intermediate reproducibilities (RSD iR ) were a bit higher, being 6.00-13.00% (for GOS 4.90-9.80%).

Conclusions
The method described is suitable for the determination of GOS in most infant formula and adult nutritional products within the concentrations typically used in such products. The precision (RSD r ) and recoveries of the method generally meet the SMPR (25). Other oligosaccharides that may be found in infant formula such as maltodextrins, FOS, polydextrose and HMO do not interfere with the analysis. The method may also be applied for assessing the proportion of the GOS that meets the definition of dietary fiber.